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. 2009 Nov 10:4:51.
doi: 10.1186/1748-717X-4-51.

The ATM and ATR inhibitors CGK733 and caffeine suppress cyclin D1 levels and inhibit cell proliferation

John P Alao  1 , Per Sunnerhagen
Affiliations

The ATM and ATR inhibitors CGK733 and caffeine suppress cyclin D1 levels and inhibit cell proliferation

John P Alao et al. Radiat Oncol. .

Abstract

The ataxia telangiectasia mutated (ATM) and the ATM- related (ATR) kinases play a central role in facilitating the resistance of cancer cells to genotoxic treatment regimens. The components of the ATM and ATR regulated signaling pathways thus provide attractive pharmacological targets, since their inhibition enhances cellular sensitivity to chemo- and radiotherapy. Caffeine as well as more specific inhibitors of ATM (KU55933) or ATM and ATR (CGK733) have recently been shown to induce cell death in drug-induced senescent tumor cells. Addition of these agents to cancer cells previously rendered senescent by exposure to genotoxins suppressed the ATM mediated p21 expression required for the survival of these cells. The precise molecular pharmacology of these agents however, is not well characterized. Herein, we report that caffeine, CGK733, and to a lesser extent KU55933, inhibit the proliferation of otherwise untreated human cancer and non-transformed mouse fibroblast cell lines. Exposure of human cancer cell lines to caffeine and CGK733 was associated with a rapid decline in cyclin D1 protein levels and a reduction in the levels of both phosphorylated and total retinoblastoma protein (RB). Our studies suggest that observations based on the effects of these compounds on cell proliferation and survival must be interpreted with caution. The differential effects of caffeine/CGK733 and KU55933 on cyclin D1 protein levels suggest that these agents will exhibit dissimilar molecular pharmacological profiles.

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Figures

Figure 1
Figure 1
Effect of CGK733, caffeine and KU55933 on cyclin D1 stability. A. MCF-7 breast cancer cells were cultured with 10 μM CGK733 for the indicated times. Total lysates were resolved by SDS- PAGE and analyzed for cyclin D1 expression. Gel loading was monitored with an antibody raised against Hsp60. B. MCF-7 cells were cultured with the indicated concentrations of CGK733 for 6 h and analyzed as in A. C. T47D breast cancer cells were treated as in B. D. MCF-7 cells were incubated with 10 μM CGK733 alone or in the presence of 40 mM LiCl or 25 μM MG132 for 6 h and analyzed for cyclin D1 expression. Gel loading was monitored with an antibody directed against α- Tubulin. E. T47D cells were treated as in D. F. MCF-7 cells were grown on coverslips and treated with 10 μM CGK733 ± 25 μM MG132 for 6 h. Cyclin D1 expression was determined by indirect immunofluorescence microscopy as described in Materials and methods. Scale bar 10 μm. G. LnCap prostate cancer cells were treated and analyzed as in D. H. MCF-7 cells were cultured with 5 mM caffeine ± 25 μM MG132 for 6 h and analyzed as in D. I. MCF-7 cells were cultured in the presence of 5 mM caffeine, 10 μM CGK733 or 20 μM KU55933 for 24 h and analyzed for RB and cyclin D1 expression. J. MCF-7 cells were cultured with the indicated concentrations of CGK733 for 24 h and analyzed as in I.
Figure 2
Figure 2
Effect of CGK733, caffeine and KU55933 on cell proliferation. A. MCF-7, T47D and MDA-MB436 breast cancer cells, LNCaP prostate cancer cells, HCT116 colon cancer cells and BALB/c 3T3 fibroblasts were cultured with the indicated doses of CGK733 for 48 h. Cell proliferation was measured as described in Materials and methods. Results represent the mean ± S.E. from three independent experiments. *, P < 0.05, compared with control; **, P < 0.005, compared with control. B. MCF-7 cells were cultured with the indicated doses of caffeine, CGK733 and KU55933 for 48 h. Cell proliferation was monitored as in A. Results represent the mean ± S.E. from three independent experiments. **, P < 0.005, compared with control. C. T47D cells were cultured with the indicated doses of caffeine, CGK733 and KU55933 for 48 h. Cell proliferation was monitored as in A. Results represent the mean ± S.E. from three independent experiments. *, P < 0.05, compared with control; **, P < 0.005, compared with control.
Figure 3
Figure 3
Caspase independent inhibition of CGK733 on MCF-7 proliferation. A. Cells were cultured with the indicated doses of CGK733 ± pan- caspase inhibitor z-VAD-fmk (40 μM) for 48 h. Cell proliferation was measured as described in Materials and methods. Results represent the mean ± S.E. from three independent experiments. B. MCF-7 cells were grown on coverslips and treated with 10 μM CGK733 or 10 μM KU55933 for 48 h. Cells were fixed and stained with DAPI as described in Materials and methods. White arrows denote condensed nuclei. Scale bar 10 μm.
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